Methods for the treatment or prevention of systemic sclerosis

ABSTRACT

The invention is in the field of molecular immunology, more in particular in the field of the prevention or treatment of autoimmune diseases, more in particular systemic sclerosis or scleroderma. The invention is based on the observation that SSC patients have an elevated plasma level of CXCL4. This was found to contribute to the pathogenesis of SSc, in particular fibrosis. When CXCL4 was neutralized in in vitro experiments, the fibrotic effects could be neutralized. This led us to conclude that SSc may be cured by reducing the plasma level of CXCL4. The invention therefore relates to a method for treatment or prevention of fibrosis in patients with scleroderma, wherein the plasma level of CXCL4 is reduced.

FIELD OF THE INVENTION

The invention is in the field of molecular immunology, more inparticular in the field of the prevention or treatment of autoimmunediseases, more in particular systemic sclerosis or scleroderma.

BACKGROUND OF THE INVENTION

Scleroderma or Systemic Sclerosis (SSc) is an autoimmune connectivetissue disorder of unknown cause characterized by microvascular injury,excessive fibrosis of the skin, and distinctive visceral involvementincluding the heart, lung, kidneys and gastrointestinal tract.Scleroderma is a progressive condition in which fibrous tissue growsabnormally, causing the skin to thicken and harden, often disfiguringand disabling patients. It affects between 5,000-10,000 new personsannually (USA) and is associated with a high morbidity and a poorprognosis. Overall, scleroderma affects an estimated 300,000 Americans,most of them women.

Forty percent of all Scleroderma patients develop at least moderaterestrictive lung disease. A high proportion of Scleroderma patients (80percent) develop lung involvement, either interstitial lung diseaseand/or pulmonary hypertension, which are the leading causes of death dueto Scleroderma. The mortality rate in Scleroderma patients with severerestrictive lung disease is about 30 percent within 10 years of onset.

The statistics underscore the need for effective treatment, preferablyat an early stage in the illness, to prevent progression to severeinterstitial lung disease. SSc is treated with oral medications to haltthe progression of disease. These drugs include cyclophosphamide, highdose prednisolone or even stem cell transplantation, all having severeside effects. In SSc, also drugs that improve circulation, promotegastrointestinal function, preserve kidney function, and control highblood pressure are given. Cyclophophamide is an anti-cancer drug thatsuppresses the immune system. This is the first drug that has beenproven to alleviate the most devastating effects of SSc. Patients takingcyclophosphamide had a significant improvement in lung function and areduction in breathlessness.

Although many research groups work on the unraveling of SScpathogenesis, to date, the exact pathways underlying the pathogenesis ofSSc remain unknown. Currently most of the research is focused on theunderlying pathways that cause fibroblast activation. A deregulatedgrowth and activation of fibroblasts has often suggested to beimplicated in SSc. However, a mode of action that is responsible forthis ongoing fibroblasts activation has not been identified.

SUMMARY OF THE INVENTION

The invention is based on the observation that SSC patients have anelevated plasma level of CXCL4. This was found to contribute to thepathogenesis of SSc, in particular fibrosis. When CXCL4 was neutralizedin in vitro experiments, the fibrotic effects could be neutralized. Thisled us to conclude that SSc may be cured by reducing the plasma level ofCXCL4. The invention therefore relates to a method for treatment orprevention of fibrosis in patients with scleroderma, wherein the plasmalevel of CXCL4 is reduced.

It was also found that CXCL4 was produced by pDCs in the bloodcirculation of patients with SSc. A method as described above istherefore preferably performed by depleting pDCs from the circulation ofa patient with scleroderma.

DETAILED DESCRIPTION OF THE INVENTION

We have found that the frequency of plasmacytoid DCs (pDCs) is markedlyincreased in SSc patients. This is illustrated in FIG. 1. In particular,the frequency of pDCs was increased in patients with the diffusecutaneous subtype of SSc. The frequency of pDCs was highest in patientssuffering from early diffuse SSc. The number of pDCs in individual SScpatients as compared to normal healthy controls is shown in FIG. 2.

We also found that plasma from SSc patients contains high levels ofCXCL4. CXCL4 is a chemokine described to bind to CXCR3, morespecifically the splice variant CXC3b, which is in contrast to CXLC9,CXCL10 and CXCL11 that bind to CXCR3a Lasagni et al J experimentalMedicine 2003. CXCL4 is known to induce apoptosis in endothelial cellsleading to endothelial cell dead. In addition, CXCL4 is known to havepotent anti-angiogenic properties and known to induce fibroblastactivation via activation of monocyte-derived dendritic cells.

CXCL4 was measured in a standard ELISA assay and the results areillustrated in FIGS. 3A. We were able to show that CXCL4 wasoverproduced by pDCs of SSc patients since elevated levels of CXCL4could also be detected in the supernatant of pDCs isolated from earlydiffuse SSc patients. This is illustrated in FIG. 3B. It was noted thatthe production of CXCL4 in pDC supernatant closely mirrored thefrequency of pDCs in plasma. Since CXCL4 was originally identified as asole platelet activation marker, we excluded the possibility thatplatelets contaminated the pDCs supernatants by measuring the β-TGcontent, which was negligible.

Hence, we conclude that CXCL4 is over-produced by pDCs in patients withSSc. This was confirmed in a whole proteome analysis using SELDI-TOF.Analysis of pDC revealed that SSc pDCs were solely committed to producea few proteins that could be identified as CXCL4 or CXCL4-precursormolecules.

We were also able to show that CXCL4 plays a role in fibroblastactivation in SSc patients. To that end, we measured the expression ofknown fibroblast activation markers TGFβR, COMP and αSMA in healthyfibroblasts co-incubated with SSc plasma. We found that SSc plasmainduced the expression of these markers for fibroblast activationsignificantly and in a dose dependent manner (FIG. 4). In addition, theplasma from healthy donors had no effect and the effect of SSc plasmawas abrogated using heparinase, a natural antagonist of CXCL4. Weconclude that an elevated level of pDCs in Ssc patients contributes tothe fibrotic process.

CXCL4 on its own was found to be unable to stimulate pDCs to producepro-inflammatory molecules; neither pDCs from normal individuals norpDCs from SSc patients produced pro-inflammatory molecules. Only in thepresence of CXCL12 specific stimulation of pDCs derived from SScpatients could be observed. This is illustrated in FIG. 6. Therein theproduction of pro-inflammatory molecules was measured in the presence ofpDCs from healthy individuals as compared to pDCs isolated from SScpatients.

pDCs from healthy controls were incubated with plasma from SSc patientsand healthy controls after which the production of pro-inflammatorymolecules was measured. The addition of plasma from SSc patients andhealthy controls had no effect. We also incubated pDCs from healthindividuals as well as from SSc patients with SSc plasma and healthycontrol plasma with and without the presence of CXCL12. We found thatonly the combination of SSc plasma and CXCL12 led to a significantlyincreased level of IL-6 and IL-12. To assure that this effect was due tothe ‘auto-activation” of pDCs by endogenous CXCL4 we added CXCL4 to thecultures of pDCs from healthy individuals with healthy control serum andCXCL12. The addition of CXCL4 to these experiments induced IL-6 andIL-12 secretion to the same levels as the combination of SSc plasma andCXCL12 (FIG. 5). In addition, the addition of heparinase fully abrogatedthe effect of SSc plasma and CXCL12 on pDCs.

Without wanting to be bound by theory, we hypothesize that CXCL12 (alsoknown as SDF-1) binds to CXCR4, thereby initiating chemo-attractiveproperties aimed at the recruitment of pDC towards the site ofinflammation. CXCL12 is known to be highly presented in the skin of SScpatients and likely to be produced by local fibroblasts. In support ofthis hypothesis it is mentioned that monocyte-derived DCs were found tobe insensitive to CXCL4 unless CXCL12 is present. It is currentlyunknown how CXCL12 sensitizes DCs to the effect of CXCL4 (Cipriani etal., Arthritis Rheum. 2006 September;54(9):3022-33).

It is known that pDCs are stimulated mostly via Toll-like receptor 9(TLR9) via which the make mainly type I interferons (IFNs). As such,pDCs are considered the main producers of this cytokine.

Challenged by the question what induces the increased CXCL4 productionby pDCs we investigated the role of Toll-like receptors (TLR). Theseexperiments showed that TLR 3, TLR7/8 and TLR 9 mediated stimulationinduced CXCL4 secretion by pDCs. Interestingly, DCs from SSc patientswere found to produce much more IFNs and IL6 than those from healthycontrols (FIG. 6). We observed a clearly increased secretion of IFNα inIcSSc patients, IdSSc patients and edSSc patients.

This shows that the potentiated TLR response by pDCs is caused by aCXCL4 dependent TLR augmentation. In support of this, neutralization ofCXCL4 led to a full abrogation of this TLR augmentation showing thatCXL4 underlies this phenomenon (FIG. 7).

To further substantiate these findings, pDCs from healthy controls wereincubated with CXCL12, CXCL4 or its combination dose dependently. Theseexperiments demonstrated that CXCL4 in combination with CXCL12 led to anaugmentation of the TLR response.

In conclusion, these observations show that CXCL4 (in conjunction withCXCL12) plays a central role in the pathogenesis of SSc. The mechanismfor that is auto-activation of pDCs resulting in a potentiated TLRresponse and further CXCL4 production.

Patients with SSc may therefore benefit from methods that remove CXCL4and/or pDCs, for instance from the blood circulation. That may beaccomplished in a number of ways, which are known in the art per se.

Patients with scleroderma may benefit from a treatment wherein theeffects of the CXCL4 produced in excess are neutralized or counteracted,for instance by removing CXCL4 from circulation. In the alternative,CXCL4 may be prevented from binding to its receptor. The inventiontherefore relates to a compound capable of binding to CXCL4 for use inthe treatment or prevention of fibrosis in patients with scleroderma.

Compounds capable of binding to CXCL4 may be compounds capable ofbinding to CXCL4 under physiological conditions.

Compounds capable of binding to CXCL4 per se are known in the art. Forinstance, fragments of the receptor or fragments of CXCL4 will besuitable compounds. In a preferred embodiment, specific antibodies maybe employed in order to remove CXCL4, for instance from the circulation.This may be accomplished by plasmapheresis.

Antibodies against CXCL4 are available in the art. They have beendescribed for instance in Vandercapellen et al., J. Leukocyte Biol. 82,1519 (2007) and are commercially available from R&D systems, 614McKinley Place NE, Minneapolis Minn. 55413.

Given the teachings of the present disclosure, it will be within theroutine skills of a person skilled in the art to design and developmaterials and methods for removing or counteracting the effects of CXCL4and/or DCs such that patients with SSc may benefit from these materialsand methods.

For that purpose, CXCL4 antagonists may advantageously employed.Antagonists for CXCL4 are known to the skilled person. As an exampleheparinase is disclosed and exemplified herein.

In another embodiment, the invention also relates to a compound capableof interfering with the in vivo production of CXCL4 for treatment orprevention of fibrosis in patients with scleroderma. Compounds capableof interfering with the in vivo production of CXCL4 per se are known inthe art. The invention therefore also relates to a method for treatmentor prevention of fibrosis in patients with scleroderma by interferingwith the in vivo production of CXCL4. Such methods may encompassinterference at the level of transcription or translation of CXCL4. Suchmethods are known in the art and may now advantageously be used in thetreatment of scleroderma. A reduction in the level of CXCL4 in apatient's body may be accomplished by a therapy in vivo. Alternatively,patients with SSc may also benefit from in vitro methods that reduce thelevel of CXCL4 in blood. Such may be done directly by applying acompound capable of binding to CXCL4 and contacting that compound withblood obtained from a patient with SSc. It may also be accomplished byreducing the number of DC that produce the CXCL4 in patients withscleroderma. This may also be accomplished in an in vitro method whereinthe previously isolated blood or plasma from a patient with sclerodermais contacted with a compound or device capable of reducing or depletingthe number of pDCs in solution. The invention therefore also relates toan in vitro method for reducing the level of CXCL4 and/or the number ofpDCs in blood obtained from a patient with scleroderma. In more detail,the invention relates to an in vitro method for reducing the level ofCXCL4 and/or the number of pDCs in a sample obtained from a patient withscleroderma, comprising the steps of:

-   -   a. Providing said sample obtained from a patient with        scleroderma    -   b. Contacting the sample with a compound capable of binding to        CXCL4    -   c. Allowing the compound to bind to CXCL4 in order to form a        complex    -   d. Removing the complex from the sample, thereby reducing the        level of CXCL4 and/or the number of pDCs.

Devices capable of reducing the level of CXCL4 and/or pDCs in blood areknown in the art. Preferably, CXCL4 and/or pDCs are removed fromcirculation, for instance using a commercially available techniqueprovided by Miltenyi (Jongbloed et al., Arthritis Res Ther.2006;8(1):R15).

Miltenyi produces and markets such a device which may advantageously beused in the new treatment according to the invention. Hence, theinvention relates to a device capable of reducing the level of CXCL4and/or pDCs in blood for the treatment or prevention of fibrosis inpatients with scleroderma.

Alternatively, a method according to the invention may also comprise astep wherein the binding of CXC4 to its receptor is blocked or otherwiseprevented or down-regulated or decreased. The invention therefore alsoprovides a method for treatment or prevention of fibrosis in patientswith scleroderma by interfering with the binding of CXCL4 to itsreceptor.

LEGEND TO THE FIGURES

FIG. 1. Increased frequency of plasmacytoid DCs in SSc patients.

Using the DC enumuration kit (Miltenyi Biotec) SSc patients showed aclearly increased frequency of plasmacytoid DCs (pDCs, BDCA2+) whereasthe frequency of myeloid DCs (mDCs, BDCA1+ or BDCA3+) was similarydistributed among groups. Especially patients with diffuse cutaneous SSchad a markedly raised frequency of pDCs that was most marked in thosehaving a disease duration shorter than 2 yrs.

FIG. 2. The increased frequency of pDCs is most pronounced in diffuseSSc. Using magnetic bead isolation of pDCs (BDCA4+ cells) it is clearthat patients with diffuse cutaneous SSc have a significantly increasedfrequency of pDCs compared to those having the limited cutaneousphenotype. In turn, patients having early diffuse (<2 yrs of diseaseduration) SSc have significantly higher frequencies of circulating pDCsthan those with late diffuse disease (>3 yrs of disease duration).

FIG. 3. CXCL4 in pDC supernatant and plasma from SSc patients ismarkedly increased. CXCL4 levels were measured using ELISA techniques inpDC supernatant (Panel A) derived from healthy controls and patientswith limited, late diffuse and early diffuse SSc. CXCL4 levels areincreased in all SSc patient s but more pronounced in patients havingdiffuse SSc especially those having early diffuse cutaneous SSc. Panel Bdisplays the CXCL4 levels in the plasma of healthy controls and SScpatients. CXCL4 levels in both pDC supernatant as well as plasma closelyreflect the frequency of pDCs in the circulation.

FIG. 4. CXCL4 directly stimulates fibroblasts.

Although unstimulated skin fibroblasts do not express high levels ofCOMP, co-incubation with SSc plasma resulted in a clear upregulation ofCOMP expression that was abrogated by heparinase, a natural CXCL4antagonist. In contrast, plasma from healthy controls did not induce theupregulation of COMP.

FIG. 5. The addition of CXCL12 to SSc pDCs induces a self-perpetuatingactivation loop. Although the IL-6 production by pDCs from healthycontrols could only be induced by the co-incubation with CXCL4+CXCL12,SSc pDCs produced large amounts of IL-6 upon the addition of CXCL12.This effect could be fully abrogated by the addition of heparinaseunderscoring the CXCL4 dependency in this system. The addition of CXCL4only did not have a significant effect suggesting the need for CXCL12 inthe CXCL4 dependent pDC activation loop.

FIG. 6. pDCs from SSc patients secrete higher levels of Type I IFNs uponTLR mediated stimulation. Upon stimulation of pDCs with ligands forTLR2, TLR3, TLR4 and TLR9, pDCs from SSc patients produce significantlymore type I IFN compared with healthy controls. More specificially, SScpatients with diffuse cutaneous disease showed a markedly increasedsecretion of type I IFN.

FIG. 7. The augmented TLR response by pDC in SSc is abrogated byneutralisation of PF4. The administration of heparinase, a natural CXCL4antagonist, fully abrogates the augmented TLR response as observed inSSc patients.

EXAMPLES Example 1 Isolation of PBMCs, Plasmacytoid Dcs and PhenotypicCharacterization

PBMCs were isolated from heparinized venous blood (100 ml) by usingdensity-gradient centrifugation over Ficoll-Paque (Amersham Bioscience).Next, plasmacytoid DCs (pDCs) were isolated using the BDCA4 magneticisolation kit from Miltenyi Biotec according to manufacturer's protocol.All procedures were carried out on ice and after the isolation of pDCs,these cells were counted using counterchambers and directly transferredto RPMI medium without any additions in a concentration of 1 millioncells/ml. After pelleting the supernatant was collected and stored at−80° C. until further analysis. The cells were then resuspended in RPMIsupplemented with 2 nM L-glutamine, 100 U/μL/ml penicillin/streptomycin(Life technologies), and 10% FCS (BioWhitacker) after which cell werebrought into culture for further analysis. Directly after isolation, apart of the BDCA4+ cells were respuspended in FACS buffer and wereanalysed for the expression of BDCA1, BDCA2, BDCA3, CD80, CD86, MHC andFcγRs applying standard flow cytometry protocols. During the firstexperiments the pDCs diamond kit from Miltenyi Biotec was exploited toreplicate our data having so-called “untouched pDCs”. In allexperiments, direct DC enumuration was performed using the DCenumuration kit from Miltenyi biotec according to the manufacturer'sprotocol.

Example 2 Measurement of Inflammatory Mediators

Supernatant from unstimulated pDCs was stored after 1 and 24 hrs ofculture. In addition, pDCs were stimulated with ligands specific forToll-like receptor 2 (TLR2, pam3Cys), TLR3 (Poly-IC), TLR4 (LPS),TLR7/TLR8 (R848) and TLR9 (CPG) for 24 hrs after which supernatant wascollected. In all these supernatants the levels of multiple inflammatorymediators (IL-8, TNF, IFNγ, IFNα, MCP-1, MIP1β, IL-6, IL-1β, IL-12.IL-23, IL-17F, IL-17A) was measured using Luminex technology aspreviously described Roelofs et al Arthritis Rheum. 2005August;52(8):2313-22., Roelofs et al Ann Rheum Dis. 2009September;68(9):1486-93. Epub 2008 Sep. 2.Wenink et al J Immunol. 2009Oct. 1;183(7):4509-20. Epub 2009 Sep. 4. To study the full proteome inpDCs, the 1 hr spontaneous supernatants were used for analysis in theSELDI-TOF. Exploiting this technique we tested 40 supernatantsoriginating from healthy controls (n=10) and SSc patients having limitedcutaneous (n=10), late diffuse (n=10) and early diffuse (n=10) diseaseas previously described (Radstake et al PLos ONE 2009).The expression ofCXCL4 and 6-TG was measured by comercially available ELISA's usedfollowing manusfacturer's protocols.

Example 3 Stimulation of Plasmacytoid DCs and Investigating theSelf-perpetuating Loop

Since the literature suggests an important role for CXCL12 in the CXCL4mediated activation of pDCs, we investigated the potential role of thisloop in SSc (vanbervliet et al J. Experimental Medicine 2003 Sep.1;198(5):823-3). To this aim, first pDCs from healthy controls and SScpatients were co-cultured with combinations of CXCL4 and/or CXCL12.During these experiments pDCs were cultured with RPMI only, RPMI/CXCL12,RPMI/CXCL12/CXCL4 and RPMI/CXCL12/heparinase. In addition, the effect ofCXCL4 on the TLR pathways was investigated by pre-incubation of pDCswith CXCL4 or the combination CXCL4/CXCL12 after whhich pDCs wereconsequetively stimulated with TLR ligands. After 24 hrs of incubation,the production of IFNα, IL-12 and IL-6 was measured by Luminex.

Example 4 CXCL4 Mediated Activation of Fibroblasts

Skin fibroblasts were isolated from healthy skin biopsies and culturedaccording t standardized protocol as described previously (Farina, AnnRheum Dis. 2009 March;68(3):435-41). When confluent, fibroblasts werereplated and cultured for an additional 24 hrs to allow attachment tothe culture plates. Subsequently, fibroblasts were incubated withcombinations of plasma from healthy controls and SSc patients, plasmaand heparinase and CXCL4/CXC12. After 48 hrs of culture, the expressionof COMP was measured by Real-time PCR as previously described (Farina,Ann Rheum Dis. 2009 March;68(3):435-41).

1. A method for treatment or prevention of fibrosis in a subject withscleroderma, the method comprising: administering to the subject acomposition comprising a compound able to bind CXCL4.
 2. The methodaccording to claim 1, further comprising removing CXCL4 and/or pDCs fromcirculation.
 3. The method according to claim 1, wherein the compound isan antagonist of CXCL4.
 4. The method according to claim 1, wherein thecompound is an antibody against CXCL4.
 5. The method according to claim1, wherein the subject suffers from the diffuse subcutaneous subtype ofscleroderma, and/or early diffuse scleroderma.
 6. In vitro method forreducing the level of CXCL4 and/or the number of pDCs in a sampleobtained from a subject with scleroderma, the method comprising:contacting the sample with a compound capable of binding to CXCL4allowing the compound to bind to CXCL4 in order to form a complex; andremoving the complex from the sample, thereby reducing the level ofCXCL4 and/or the number of pDCs in the sample.
 7. The method accordingto claim 6, wherein the sample is blood, plasma, or serum.
 8. The methodaccording to claim 6, wherein the compound is an antibody against CXCL4.9. A device capable of reducing the level of CXCL4 and/or pDCs in asample the device comprising: an antibody able to specifically bind toCXCL4.
 10. The method according to claim 2, wherein the compound is anantagonist of CXCL4.
 11. The method according to claim 2, wherein thecompound is an antibody against CXCL4.
 12. The method according to claim3, wherein the compound is an antibody against CXCL4.
 13. The methodaccording to claim 7, wherein the compound is an antibody against CXCL4.14. A method of reducing fibrosis in a subject in need thereof, themethod comprising: reducing CXCL in the subject; or reducing CXCL4activity in the subject.